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Stargardt’s disease is the most common inherited macular dystrophy causing progressive impairment of central vision. It is associated with mutations in the ABCA4 gene, encoding photoreceptor cell-specific ATP-binding cassette transporter 4 protein. The disease is inherited in an autosomal recessive fashion. The ABCA4 protein is predominantly expressed in the retina, where it is involved in transport of N -retinylidene-PE. Absence of ABCA4 results in the failure to clear these toxic substances, resulting in the loss in photoreceptor cells. A large number of disease-causing mutations have been found in ABCA4 . c.5461-10T>C is the third most frequent ABCA4 mutation, and causes a severe form of Stargardt’s disease. This mutation is located in intron 38, and leads to skipping of exon 39, or of exon 39 and exon 40 in the mRNA. This aberrant splicing pattern results in reduced ABCA4 protein level. QRX-1011 targets the ABCA4 pre-mRNA and results in the retention of exon 39 and exon 40, leading to the production of a mature wild type mRNA and protein.
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Clinical Presentation of Stargardt’s Disease
The most common symptom of Stargardt’s disease is slow loss of central vision in both eyes. Onset of the disease is typically in childhood or young adulthood. Patients notice gray, black, or hazy spots in the center of their vision, have reduced light adaptation with increased light sensitivity, and some patients also experience color blindness as the disease progresses. Most patients with Stargardt’s disease will progress to legal blindness or worse and may also suffer constriction of the visual field as they age.
It is estimated there are 7,000 Stargardt’s disease patients in the Western world with the c.5461-10T>C mutation in ABCA4 .
Approaches for the treatment of Stargardt’s disease
Currently, there is no treatment available for Stargardt’s disease. Patients are often advised to wear eyeglasses or sunglasses that block UV light to reduce the possibility of additional eye damage caused by the sun and to avoid taking vitamin A supplements, but these measures do not prevent the progression of the disease. As Stargardt’s disease due to the ABCA4 c.5461-10T>C mutation is inherited in an autosomal recessive manner, the condition may be amenable to gene therapy approaches where the complete loss of ABCA4 function is complemented by simple gene replacement.
QRX-1011 for the treatment of Stargardt’s disease
QRX-1011 is a first-in-class single-stranded oligonucleotide designed to treat vision loss caused by the specific c.5461-10T>C mutation in the ABCA4 gene which leads to a splicing defect. Using an antisense oligonucleotide which modulates splicing of the mRNA, QRX-1011-mediated correction in the mRNA level leads to inclusion of the deleted exons and formation of functional, wild type ABCA4 protein which will potentially stop and perhaps reverse the progression of the disease.
Dystrophic Epidermolysis Bullosa (DEB)
Epidermolysis bullosa (EB) is a rare genetic disorder, primarily manifesting as a debilitating disease of the skin and mucosal membranes. It is characterized by mechanical fragility of epithelial tissues, blister formation, scarring and, in some subtypes, involvement of multiple other organs. EB is classified into four main subtypes, namely EB simplex (EBS), junctional EB (JEB), dystrophic epidermolysis bullosa (DEB), and Kindler Syndrome (KS). The four main EB subtypes are distinguished by the level of the skin at which blisters develop.
In DEB, the outer layer of the skin, the epidermis, separates from the inner layer, the dermis. This separation renders the skin fragile and causes severe blistering and has downstream effects such as wound infection, scarring, and SCC (squamous cell carcinoma). All mucosal membranes are affected in DEB, therefore blistering is not limited to the skin, but is also present in the mouth, esophagus and downstream intestines.
DEB is usually a chronic, seriously debilitating disease with a shortened life expectancy due to malnutrition, infections, and malignancies.
The disease is caused by mutations in the COL7A1 gene. This gene is responsible for the production of a protein called collagen type VII (also referred to as C7), which is a major component of the anchoring fibril located below the basement membrane that normally links the epidermis and the dermis together. DEB causing mutations occur more often in certain parts of the gene. One of those parts is exon 73.
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DEB Prevalence and Diagnosis
DEB is a genetic disease that in some cases is inherited as an autosomal dominant (DDEB) and in others as an autosomal recessive trait (RDEB). The prevalence of DEB could differ across countries due to founder effects and differences in ethnic composition. While spatial variations, compounded with the scarcity of available data, make accurate calculations difficult, the estimated number of DEB patients in the Western world is approximately 6,000 of which approximately 2,000 have a mutation in exon 73.
Diagnostic testing for DEB is based on the identification of the level of skin cleavage via immunofluorescence antigen mapping with C7 specific antibodies and/or determination of anchoring fibrils using transmission electron microscopy.
Approaches for the Treatment of DEB
Currently, no disease modifying treatment is available for DEB. Palliative treatment is the only treatment available for DEB patients and constitutes a time-consuming daily activity. Palliative treatment primarily consists of care of (new) blisters by puncturing and draining to prevent further spread from fluid pressure, wound management to prevent infections, prevention of skin trauma to avoid new blister formation, and pain and itch relief.
QR-313 for the treatment of DEB
QR-313 is designed to specifically target mutations in exon 73 of the COL7A1 gene. QR-313 binds to a specific sequence in the COL7A1 pre-mRNA, thereby excluding exon 73 from the mature mRNA. This leads to a shortened version of the C7 protein that is functional in the formation of anchoring fibrils.
Because of the exon skipping approach, QR-313 is not specific to a single mutation but instead targets any mutation contained in exon 73.
Pre-clinical evidence for QR-313
Clinical development of QR-313 focuses on topical delivery in the wounded skin of patients, with the aim to improve wound healing and reduce skin fragility. Therefore, we formulate QR-313 into a hydrogel for wound application that can be incorporated in the standard of care of patients.
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Activity of QR-313 in cells and human skin equivalents
The activity of QR-313 was investigated in 3 different in vitro test systems; cell lines, primary cells, and human skin equivalents (HSEs). HSEs are composed of both a dermal layer containing fibroblasts and an epidermal layer containing keratinocytes. The keratinocytes are fully differentiated to form all the different layers in the epidermis, including the stratum corneum. The culturing of HSEs is done at the air-liquid interface and therefore mimics the human situation. Moreover, by removing the epidermis from a portion of the skin equivalent, the blistering phenotype of DEB can be modeled.
Experiments have shown
The effect of QR-313 was assessed in HPFs from an RDEB patient, which contain one pathogenic mutation in exon 73 of COL7A1 and another pathogenic mutation on the other allele. Results showed efficient skipping of exon 73 from COL7A1 mRNA after 24 hours, and an increasing efficiency after 48 hours, with a near absence of the full length transcript that contains exon 73. In a subsequent set of experiments also lower concentrations were tested in RDEB HPFs as well as wild type HPFs. The results showed a clear dose response for QR-313 with increasing exon skip percentages in both RDEB and wild type HPFs. With the high doses of QR-313 a median skip of 77% for wild type and 87% for RDEB HPFs is reached.
Sequence analysis on the 150 bp product demonstrates removal of the complete exon 73 from the mRNA. This provides further evidence that QR-313 acts via its intended mode of action in human cells and efficiently skips exon 73 in the COL7A1 mRNA.
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